Tbe HIV-1 tat gene product is a transactivator of viml gene expression. Tat promotes transcription from the HIV- I LTR, resulting in increased synthesis of viral MRNA. Several lines of evidence suggest that the Tat activation responsive region (terrned TAR), within the HIV- I LTR, is recognized as a stem-looped nascent RNA. Although Tat has been demonstrated to bind to TAR RNA in vitro, the requirements for Tat binding are not sufficient to account for the multiple sequence and structural requirements for transactivation. Our current model of viml gene activation by Tat involves the interaction of Tat with Tar RNA in combination with one more cehular factor(s). In order to further understand the complex mechanism of HIV gene regulation, we will investigate the protein components (Tat and cellular proteins) which participate in the transactivation pathway using genetic and biochemical approaches. In order to further investigate the interaction of Tat with the TAR sequence, we will take advantage of the difference in specificity between HIV-1 and the more closely related HIV-2 and SIV Tat proteins. Using chimeric HIV-L/SIV and HIV-l/HIV-2 tat constructs in transactivation assays, a domain of Tat responsible for LTR specificity will be identified. Mutagenesis within this region will further define the amino-acid residues which specify TAR recognition. The ability of HIV-1 Tat to recognize a smaller target relative to the HIV-2 and SFV proteins, may permit more promiscuous interactions with host gene regulation and may contiibute to the pathogenesis in AIDS. Tbe Tat transactivation pathway involves the function of one or more cellular TAR RNAbinding proteins. Several groups have idenfified TAR RNA-binding activifies in nuclear extracts. Marciniak et al.(1990) have identified a 68 kDa polypeptide (in HeLa cells) which increases transactivation by Tat in cell-free extracts. Our aim is to identify and characterize TAR RNA-binding polypeptides which function, in concert with Tat, to promote HIV- I gene expression in target cells. We have identified a 46 kDa polypeptide in H9 nuclear extracts which recognizes TAR RNA in UV cross-linking assays. We propose to purify this polypeptide and determine its role in HIV- I gene expression. Given the essential role of Tat in HIV-1 viral replication, the Tat activation pathway is an attractive control point for the development of therapeutic moclalities. In addition to increasing our basic understanding of FEV gene regulation, the identification of one or more cellular factor(s) involved in Tat activation would open additional avenues of investigation toward antiviral therapies in AIDS.